Trace-Level Determination of Acrylonitrile Generated in the Manufacturing Process of Oligonucleotides by Static Headspace Gas Chromatography with an Electron Impact(+) Mass Detector

A static headspace capillary gas chromatography (HS-GC) method featured with a short run time, a wide calibration range, and a diluent with strong buffering and stabilizing capability has been developed for the quantitative analysis of trace levels of acrylonitrile, a known genotoxic impurity and potential byproduct of oligonucleotide manufacturing. This GC method was achieved on a commercially available fused silica capillary column, and the total run time including GC oven temperature re-equilibration is 20 min. The method employs a split injection (5:1) and a programmed temperature ramp and was qualified in terms of specificity, accuracy, repeatability/precision, quadratic calibration range, limit of quantitation (0.40 ppm), limit of detection (0.10 ppm), solution stability, and robustness. The method was demonstrated to be quadratic over the range of four orders of magnitude from 0.40 to 4000 ppm with a correlation coefficient greater than 0.99 with average recoveries between 86 and 101%. Method performance was also evaluated using sample matrices of different pH values and compositions, and the method was demonstrated to be suitable as a platform test method for the determination of acrylonitrile in the manufacturing process of oligonucleotides. Finally, the degradation kinetics of acrylonitrile in backbone deprotection (1:1 acetonitrile/triethylamine) and ammonolysis (30% NH3 aqueous) matrices was assessed. Acrylonitrile was demonstrated to degrade quantitatively and quickly in the ammonolysis sample matrix.

Automated summary

A static headspace capillary gas chromatography (HS-GC) method has been developed for the quantitative analysis of trace levels of acrylonitrile, featuring a short run time, wide calibration range, and a diluent with strong buffering and stabilizing capability. The method was validated for specificity, accuracy, repeatability/precision, calibration range, limits of quantitation and detection, solution stability, and robustness, showing quadratic performance over a wide range of concentrations. This method is deemed suitable as a platform test method for acrylonitrile determination in oligonucleotide manufacturing processes.